Plk1 and CK2 Act in Concert to Regulate Rad51 during DNA Double Strand Break Repair

Summary Homologous recombination (HR) plays an important role in the maintenance of genome integrity. HR repairs broken DNA during S and G2 phases of the cell cycle but its regulatory mechanisms remain elusive. Here, we report that Polo-like kinase 1 (Plk1), which is vital for cell proliferation and is frequently upregulated in cancer cells, phosphorylates the essential Rad51 recombinase at serine 14 (S14) during the cell cycle and in response to DNA damage. Strikingly, S14 phosphorylation licenses subsequent Rad51 phosphorylation at threonine 13 (T13) by casein kinase 2 (CK2), which in turn triggers direct binding to the Nijmegen breakage syndrome gene product, Nbs1. This mechanism facilitates Rad51 recruitment to damage sites, thus enhancing cellular resistance to genotoxic stresses. Our results uncover a role of Plk1 in linking DNA damage recognition with HR repair and suggest a molecular mechanism for cancer development associated with elevated activity of Plk1.

Recombinant full-length FLAG-Nbs1 was a kind gift from Dr Tanya Paull, and recombinant Plk1 and CK2 protein were purchased from Abcam and New England BioLabs respectively. pCBASceI (Richardson et al., 1998) was obtained from Addgene (gene ID: 26477). Genes encoding AAVS1 targeting zinc-finger domains are synthesised from GeneArt according to the previously described amino acid sequence (Hockemeyer et al., 2009), and cloned into pZFN1 and pZFN2 (Sigma, kind gifts from Prof. Peter Cook) at Acc65I-BamHI sites. To generate pZDonor-AAVS1-GFP, a GFP gene was PCR cloned into pZDonor-AAVS1-Puromycin (Sigma) at BglII-NcoI sites.
Antibodies were affinity purified using phosphopeptide columns prepared with SulfoLink Kit (Pierce).
After elution with ImmunoPure Gentle Ag/Ab Elution Buffer (Pierce), antibodies were dialysed against TBS and contaminating non-phosphospecific antibody was affinity depleted by passing through a column cross-linked with non-phosphopeptide (NH2)-CEANADTSVEEE-(COOH). The eluted phospho-specific antibodies were then enriched by dialysis against TBS containing 50% glycerol.

Mass Spectrometry Analysis for Identification of Phosphorylation Sites
Polyacrylamide gel slices (1-2 mm) containing the purified proteins were prepared for mass spectrometric analysis using the Janus liquid handling system (PerkinElmer, UK). Briefly, the excised protein gel pieces were placed in a well of a 96-well microtitre plate and destained with 50% v/v acetonitrile and 50 mM ammonium bicarbonate, reduced with 10 mM DTT, and alkylated with 55 mM iodoacetamide. After alkylation, proteins were digested with 6 ng/µL Trypsin (Promega, UK) overnight at 37°C. The resulting peptides were extracted in 2% v/v formic acid, 2% v/v acetonitrile.  (Perkins et al., 1999). Database search parameters were set with a precursor tolerance of 5 ppm and a fragment ion mass tolerance of 0.8 Da. One missed enzyme cleavage was allowed and variable modifications for oxidized methionine, carbamidomethyl cysteine, pyroglutamic acid, phosphorylated serine, threonine and tyrosine were included. MS/MS data were validated using the Scaffold programme (Proteome Software Inc., USA) (Keller et al., 2002). All data were additionally interrogated manually.

Analysis of Cell Cycle Distribution by Flow Cytometry
U2OS cells were pulse labeled with 10 μM bromodeoxyuridine (BrdU) (Sigma-Aldrich) for 30 min prior to harvesting by trypsinisation. Cells were washed in PBS and fixed with ice cold 70% ethanol for 30 min on ice. Fixed cells were washed in PBS and DNA was denatured with 2 N HCl containing 0.5% Triton X-100 for 30 min at room temperature, followed by neutralisation with 0.1M Na 2 B 4 O 7 at pH 8.5.

Immunofluorescence Microscopy
Cells were seeded at a density of 1 x 10 5 cells in 12-well plates containing coverslips 24 hours after siRNA transfection. After overnight incubation, cells were irradiated with 4 Gy, and coverslips were recovered after 2.5 hours. The cells were pre-extracted for 1 min in pre-extraction buffer (80 mM NaCl, 3 mM MgCl 2 ) and fixed with 4% paraformaldehyde in PBS, followed by permeabilisation with 0.5% Triton X-100 in PBS. After blocking in antibody dilution buffer (1% BSA, 0.2% cold fish skin gelatin and 0.05% Triton X-100 in PBS), primary antibody against Rad51 and -H2AX were applied.

Small Interfering RNAs
Following small interfering RNAs were synthesized from Sigma-Aldrich, and a mixture of two siRNA was used to down regulate indicated gene product.
(B) Similarly, Rad51 NTD variants with S14 substitution were phosphorylated with CK2, and its interaction with Nbs1 was detected by far-western blotting. Consistent with the altered CK2-mediated phosphorylation of Rad51 S14 variants shown in Figure 3D, increased Nbs1 interactions with CK2 phosphorylated S14D or S14E NTD, but reduced interaction with S14A NTD, were detected. Figure S4. Phenotypes of Cells Expressing Rad51 Variants, Related to Figure 6 (A) Stable expression of S14 Rad51 variants in U2OS cells was confirmed after down-regulating endogenous Rad51 with siRNA targeting the 3' UTR. Lamin was used as a loading control.
(B) Cell cycle profiles of S14 Rad51 variant expressing cells. Cells were pulse-labeled with 5bromodeoxyuridine to detect cells actively replicating DNA, and DNA contents were detected with PI staining using flow cytometry.
(C and D) Cells treated with siRad51 (C) or siBRCA2 (D) were irradiated at 4 Gy, and cells containing more than twenty Rad51 foci at indicated time after IR was counted.
(E) Five hundred cells with down-regulated endogenous Rad51 or BRCA2 by siRNA were seeded, and the resulting colonies were counted after two weeks. There was no significant difference in clonogenic survival among cells expressing different Rad51 variant. Error bars s.d. (n=3).
(F) An example of HR assay depicted in Figure 6H, showing a representative flow cytometric profile of U2OS-SCR18 cells exogenously expressing wild-type Rad51.
(G and H) Examples of DSB-mediated gene targeting assay depicted in Figure 6J, showing a representative flow cytometric profile of U2OS cells exogenously expressing wild-type Rad51 (G) or EUFA423 cells exogenously expressing wild-type Rad51 (H).